镇江香醋固态发酵醋醅中微生物总DNA提取方法比较  

Comparison of Total Microbial DNA Extraction Methods from Solid-culture of Zhenjiang Vinegar

作　　者：倪峥飞[1,2] 许伟[2] 窦文芳 许泓瑜 许正宏[1,2] 

机构地区：[1]江南大学医药学院制药工程实验室,无锡214122 [2]江南大学食品科学与技术国家重点实验室,无锡214122

出　　处：《微生物学报》2010年第1期119-125,共7页Acta Microbiologica Sinica

基　　金：国家863前沿探索项目(2007AA02Z203);国家十一五科技支撑计划(2006BAD27B09-3;2008BAI63B06);教育部新世纪人才支持计划(NCET-07-0380);江南大学食品科学与技术国家重点实验室目标导向课题(SKLF-MB-200801)~~

摘　　要：【目的】为了更加全面地分析我国传统固态发酵过程中微生物群落的多样性和演替情况,本文以镇江香醋固态发酵为例,对比研究了11种不同的总DNA提取法对醋醅中总DNA提取的影响。【方法】使用紫外分光光度计法和荧光定量PCR(Realtime Quantitative PCR)测定了不同提取方法得到的醋醅样品总DNA的产量与纯度,采用变性梯度凝胶电泳(DGGE)法对固态发酵中细菌和真菌的多样性进行了分析。【结果】醋醅总DNA得率最高可达93.2±1.5μg/g干醅,细菌总数最高达到1.73×1013 copies.(g干醅)-1,真菌总数最高达到6.49×1012 copies.(g干醅)-1。不同的提取方法对DGGE结果有明显的影响,6种基于SDS裂解的方法所获得的条带较多。【结论】结果表明,液氮研磨+溶菌酶+SDS高盐抽提法(方法3)为最优的醋醅总DNA提取方法。[ Objective] To analyze the diversity and succession of microbes during traditional solid-state fermentation. Taking Zhenjiang vinegar for example, we compared 11 methods for extracting genomic DNA from microbes in solid-culture of Zhenjiang vinegar. [Methods] The yield and purity of the DNA were measured by ultraviolet spectrophotometer and real-time PCR. The bacterial and fungal diversity during solid-state fermentation was analyzed by denaturing gradient gel electrophoresis （DGGE）. [ Results] The highest extraction yield of total DNA was 93.2 ±1.5μg/g dried culture. The quantity of bacteria and fungi reached to the maximum of 1.73 ×10^13 copies · （ gram dried culture） -1 and 6.49 ×10^12 copies· （gram dried culture） -1, respectively. The community patterns revealed by DGGE were noticeably influenced by the DNA extraction methods. Hereinto, six methods of SDS-based DNA extraction resulted in the larger number of DNA bands. [ Conclusion] The results showed that the most effective method was SDS high salt extraction combined with grinding in liquid nitrogen and lysozyme lysis.

关 键 词：食醋固态发酵 DNA提取 荧光定量PCR 变性梯度凝胶电泳 

分 类 号：Q93[生物学—微生物学]